Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease, motor neuron disease). With increasing age, insoluble forms of mutant SOD1 progressively accumulate in neural tissues of human ALS patients and in spinal cords of transgenic mice expressing these polypeptides, suggesting that SOD1- linked ALS is a protein misfolding and aggregation disorder. Understanding the molecular basis for how the pathogenic mutations give rise to SOD1 folding intermediates that are prone to aggregation is therefore of keen interest. A critical step on the folding pathway occurs when the nascent SOD1 polypeptide is posttranslationally modified by the copper chaperone for SOD1 (CCS), a helper protein that inserts the catalytic copper cofactor and oxidizes the SOD1 intrasubunit disulfide bond. CCS recognizes and binds to nascent, but not mature SOD1, suggesting that the newly translated form exists in a conformation distinct from the mature form. Recent studies reveal that pathogenic SOD1 proteins coming from aggregates isolated from cultured cells and from the spinal cords of transgenic mice tend to be metal-deficient and/or lacking the disulfide bond. These observations suggest the hypothesis that the disease-causing mutations may enhance levels of SOD1 folding intermediates by hindering CCS-mediated SOD1 maturation. The experiments outlined in this project are designed to probe the structure and dynamics of nascent SOD1 and to examine its interaction with CCS. Successful completion of the Aims contained herein will address the following questions: 1) What are the structural determinants that are responsible for the recognition of nascent SOD1 by CCS? 2) Is the functional SOD1/CCS complex heterodimeric, heterotetrameric, or some other higher order oligomer? 3) What are the structural details of the nascent SOD1/CCS complex in three dimensions? 4) What factors govern the interconversion of the nascent and mature SOD1 conformations in the absence of CCS? 5) How do the ALS mutations affect the interconversion of the nascent and mature conformations? Answers to these questions are prerequisites for the design of therapeutic agents aimed inhibiting pathogenic SOD1 aggregation in ALS. 1